Medium composition for culturing animal cells for producing recombinant extracellular matrix protein and method of using the same

ABSTRACT

Provided are a medium composition for culturing animal cells for producing a recombinant extracellular matrix protein, a method of producing the recombinant extracellular matrix protein with high purity, and a method of assaying a monomer of the recombinant extracellular matrix protein.

TECHNICAL FIELD

The present disclosure relates to a medium composition for culturinganimal cells for producing a recombinant extracellular matrix protein, amethod of producing the recombinant extracellular matrix protein withhigh purity, and a method of assaying a monomer of the recombinantextracellular matrix protein.

BACKGROUND ART

The extracellular matrix (ECM) is a non-cellular component in livingthings, formed by various substances that are secreted out of cells, andis present within all tissues and organs of living organisms. Theextracellular matrix performs various biological functions such ascell-to-cell adhesion and physical support, as well as celldifferentiation and growth, and intercellular signaling and regulation,etc. Since each tissue and organ of a multicellular organism haveindependently evolved according to their characteristics, the componentsand functions of the extracellular matrix also vary depending on thetype of tissue and cell.

Basically, the extracellular matrix consists of water, proteins, andpolysaccharides. Among them, extracellular matrix proteins areself-assembled into a molecular scaffold by adjusting biomechanicalproperties and composition according to functions of the correspondingtissue, and most extracellular matrix proteins are expressed in a traceamount in the tissue, and function by forming multiple bonds in amodular form. Due to these structural features, studies regardingmass-production and functions through recombination of extracellularmatrix proteins have faced numerous technical challenges. Generally,proteins extracted from animal tissues are used, but the amounts of theextracted proteins are very small. Therefore, to mass-produceextracellular matrix proteins using desired amino acid sequences, anecessity of having more appropriate and cost-effective productionsystems has been raised.

Meanwhile, it has been reported that hyaluronan and proteoglycan link(HAPLN) protein among extracellular matrix proteins plays a role instabilizing aggregates of hyaluronic acid and proteoglycan in theextracellular matrix and is involved in cell-to-cell adhesion. There area total of four types of HAPLN proteins depending on tissues or organsin vivo where they are expressed; HAPLN1, HAPLN2, HAPLN3, and HAPLN4. Itis known that their functions and roles in each tissue or organ are notsubstantially distinct.

Korean Patent No. 10-1897340 discloses a pharmaceutical composition forimproving skin elasticity or wrinkles, the pharmaceutical compositionincluding HAPLN1 protein as an active ingredient, Korean PatentPublication Nos. 10-2019-0024727 and 10-2020-0104831 disclose acomposition for regenerating cartilage and a composition for treatingcartilage-related diseases, each composition including HAPLN1 protein asan active ingredient. Recently, Korean Patent No. 10-2166453 discloses acomposition for treating lung diseases, the composition including HAPLN1protein as an active ingredient. As described, HAPLN proteins areexpected to provide useful functions to humans. Accordingly, formass-production of recombinant HAPLN proteins, it is necessary to studyculture, isolation, purification, and monomer assay methods. However, amethod of mass-producing recombinant HAPLN proteins has not yet beenstudied.

DISCLOSURE OF INVENTION Technical Problem

There is provided a medium composition for culturing animal cells forproducing a recombinant extracellular matrix protein.

There is provided a method of producing the recombinant extracellularmatrix protein with high purity.

There is provided a method of assaying a monomer of the recombinantextracellular matrix protein.

Solution to Problem

An aspect provides a medium composition for culturing animal cells forproducing a recombinant extracellular matrix protein, the mediumcomposition including a copper compound.

The term “extracellular matrix (ECM) protein” refers to a proteinpresent in the extracellular matrix. Exemplary types of the ECM proteininclude collagen, elastin, fibronectin, laminin, vitronectin, tenacin,hyaluronan and proteoglycan link protein (HAPLN), etc., but are notlimited thereto.

The term “hyaluronan and proteoglycan link protein (HAPLN)” is alsocalled hyaluronic acid and proteoglycan link protein. There are a totalof four types of HAPLN proteins depending on tissues or organs in vivowhere they are expressed, and the types thereof include HAPLN1, HAPLN2,HAPLN3, and HAPLN4. An amino acid sequence of the HAPLN protein isdescribed in HAPLN1, for example, human HAPLN1 Accession No. NP_001875,or mouse HAPLN1 Accession No. NP_038528, etc., but is not limitedthereto.

The term “recombinant ECM protein” refers to a protein obtained byexpressing DNA in cells, the DNA encoding an ECM protein produced usinga genetic recombination method. The genetic recombination may beperformed according to a common method in the art.

In one embodiment, the recombinant extracellular matrix protein may becollagen, elastin, fibronectin, laminin, vitronectin, tenascin, orHAPLN, but is not limited thereto.

The term “recombinant HAPLN protein (rHAPLN)” refers to a proteinobtained by inserting a polynucleotide sequence encoding the HAPLNprotein into a vector to construct a recombinant vector, introducing therecombinant vector into a host cell, and expressing the recombinantvector in the cell.

In one embodiment, the recombinant HAPLN protein may be any one proteinselected from the group consisting of HAPLN1, HAPLN2, HAPLN3, andHAPLN4.

The term “recombinant human HAPLN protein (recombinant human HAPLN,rhHAPLN)” refers to a protein obtained by inserting a polynucleotidesequence encoding the human HAPLN protein into a vector to construct arecombinant vector, introducing the recombinant vector into a host cell,and expressing the recombinant vector in the cell.

The recombinant extracellular matrix protein may be a protein derivedfrom a human body or an animal. In one embodiment, the recombinantextracellular matrix protein may be a protein derived from a human body.The term “animal cells for producing the recombinant ECM protein” or“animal cells producing the recombinant ECM protein” refers to animalcells into which the recombinant vector is introduced to produce therecombinant ECM protein.

The animal cells are not limited to the type thereof, as long as theyare cells capable of producing the recombinant ECM protein. The animalcells may be selected from the group consisting of Chinese Hamster Ovary(CHO), VERO, Baby Hamster Kidney (BHK), HeLa, NiH 3T3, Madin-DarbyCanine Kidney (MDCK), WI38, Human Embryonic Kidney (HEK), hybridoma, andNSO cells. The animal cells may be CHO cells or CHO cell variants. TheCHO cells may be CHO-K1, CHO-DXB11, CHO-DG44, CHO-S, or CHO-Pro minuscells.

The term “copper compound” refers to a compound of copper, and copperwith the oxidation state of +1, +2, or +3 is known.

The type of the copper compound included in the medium composition isnot limited. The copper compound may be a copper (I) compound, a copper(II) compound, or a copper (III) compound. The copper compound may becopper oxide (Cu₂O), copper chloride (CuCl₂), copper nitrate (Cu(NO₃)₂),copper II oxide (CuO), copper sulfide (CuS), or copper sulfate (CuSO₄),but is not limited thereto.

The medium composition may include the copper compound at aconcentration of more than about 20 µM, about 30 µM or more, about 40 µMor more, about 50 µM or more, for example, more than about 20 µM toabout 1,000 µM, about 30 µM to about 1,000 µM, about 40 µM to about1,000 µM, about 50 µM to about 1,000 µM, about 50 µM to about 900 µM,about 50 µM to about 800 µM, about 50 µM to about 700 µM, about 50 µM toabout 600 µM, about 50 µM to about 500 µM, about 50 µM to about 400 µM,about 50 µM to about 300 µM, about 50 µM to about 200 µM, about 50 µM toabout 100 µM, about 50 µM to about 90 µM, about 50 µM to about 80 µM,about 50 µM to about 70 µM, about 50 µM to about 60 µM, or about 50 µM.The medium composition may separate, into monomers, the ECM protein thatbinds with multiple bonds in a modular form by including the coppercompound at a concentration of more than about 20 µM, particularly,about 50 µM or more. When the concentration of the copper compound isabout 20 µM or less, formation of protein multimers may be increased,and thus a protein production amount may be decreased. Therefore, byincluding the copper compound at a concentration of more than about 20µM, particularly, about 50 µM or more, the medium composition may reduceformation of recombinant ECM protein multimers. Consequently, when themedium composition is used, the ECM protein may be separated intomonomers, and thus it is possible to solve the problem of difficulty inmass-production due to the structural feature in which the ECM proteinis combined in a modular form after being expressed at a trace amount.

The medium composition may further include general medium componentsused for culturing animal cells. The general medium components arecomponents needed for culturing animal cells to obtain the recombinantprotein, and any known or commercially available components may be used.

The medium composition may further include an additional additive. Themedium composition may further include one or more additives of dimethylsulfoxide (DMSO); glycerol; poloxamers such as poloxamer 188; EDTA;polysorbates such as polysorbate 80; cysteine; glutathione (GSH);glutathione disulfide (GSSG); and magnesium chloride (MgCl₂), but is notlimited thereto.

In one exemplary embodiment, it was confirmed that when copper sulfatewas added to a medium during culturing animal cells producing arecombinant human HAPLN1 protein, it exhibited the excellent effect ofreducing formation of protein multimers, and thus the protein productionamount was increased. Therefore, the medium composition including thecopper compound may be usefully applied to obtaining a large amount ofthe recombinant ECM protein from animal cells.

In one embodiment, the medium composition may be a medium compositionfor culturing animal cells for mass-producing the recombinant HAPLNprotein, specifically, a medium composition for culturing animal cellsfor mass-producing the recombinant human HAPLN protein, and morespecifically, a medium composition for culturing animal cells formass-producing the recombinant human HAPLN1 protein.

In the medium composition, the recombinant extracellular matrix proteinmay be a monomer. The “monomer” is used interchangeably with a “monomerprotein”, and refers to one of proteins constituting a multi-proteincomplex. A complex of two or more polypeptides is referred to as a“multimer” or an “oligomer”, a complex of two polypeptides is referredto as a dimer, a complex of three polypeptides is referred to as atrimer, and a complex of four polypeptides is referred to as a tetramer.Since the medium composition including the copper compound has theeffect of reducing formation of the multimers of the recombinant ECMprotein, it may be used for producing the monomer of the recombinant ECMprotein. In other words, the medium composition including the coppercompound may separate, into monomers, the ECM protein that binds withmultiple bonds in a modular form. Therefore, the medium composition maybe a medium composition for culturing animal cells for producing themonomer of the recombinant ECM protein.

Another aspect provides a method of producing the recombinantextracellular matrix protein with high purity, the method including

(1) culturing animal cells producing the recombinant ECM protein in themedium composition according to an aspect and obtaining a culturemedium; and

(2) isolating and purifying the recombinant ECM protein from the culturemedium.

The medium composition, the recombinant ECM protein, and the animalcells are the same as described above.

According to the method of producing the recombinant ECM protein, therecombinant ECM protein with high purity may be mass-produced.Therefore, the method may be a method of mass-producing the recombinantECM protein, specifically, a method of mass-producing the recombinantHAPLN protein, more specifically, a method of mass-producing therecombinant HAPLN1 protein, and most specifically, a method ofmass-producing the recombinant human HAPLN1 protein.

The culturing of (1) may be performed using a method widely known in theart. For example, the culturing may be performed by fed-batch culture,continuous culture, batch culture, etc. In one embodiment, the culturingof (1) may be fed-batch culture.

The term “fed-batch culture”, which is a culture method ofintermittently feeding a medium, refers to a culture method capable offreely controlling an amount of a substrate to be fed, because thesubstrate in the culture medium is added at an appropriate rate withoutdischarge.

The term “continuous culture” refers to a culture method, in which a newnutrient medium is continuously fed, and at the same time, the culturemedium containing cells and products is continuously removed.

The term “batch culture”, which is a method of continuing culture untilall of raw material substrates initially fed are consumed, refers to aculture method, in which concentrations of a substrate, metabolites, andcells are continuously changed over time.

The culturing of (1) may be performed for about 5 days to about 15 days,about 5 days to about 13 days, about 8 days to about 15 days, about 8days to about 13 days, about 10 days to about 15 days, about 10 days toabout 13 days, about 11 days to about 15 days, or about 11 days to about13 days, but is not limited thereto.

In (1), the copper compound may be added once or twice or more to themedium composition during cell culture.

In (1), the culturing may be performed by adding the copper compound tothe medium composition before cell culture. For example, the coppercompound may be added to the medium composition on day 0 of cellculture.

In the method of producing the recombinant ECM protein, the recombinantECM protein may be a monomer. Therefore, the method may be a method ofproducing a monomer of the recombinant ECM protein, specifically, amethod of producing a monomer of the recombinant HAPLN protein, morespecifically, a method of producing a monomer of the recombinant HAPLN1protein, and most specifically, a method of producing a monomer of therecombinant human HAPLN1 protein.

(2) may include performing chromatography. The chromatography may be anyone or more selected from the group consisting of affinitychromatography, anion exchange chromatography, cation exchangechromatography, hydroxyapatite chromatography, reversed-phasechromatography, size exclusion chromatography, mixed modechromatography, and hydrophobic interaction chromatography.

(2) may include performing anion exchange chromatography.

The term “ion exchange chromatography (IEC)” refers to a method ofseparating and analyzing sample ions using an affinity difference for astationary phase by performing a reversible ion exchange between thestationary phase and a mobile phase using an ion exchanger in thestationary phase.

The term “anion exchange chromatography (AEX)” is a type of ion exchangechromatography, and uses an anion exchanger having a cationic functionalgroup such as an amino group, etc.

The anion exchange chromatography may include pre-equilibration,equilibration, sample loading, washing, and elution.

The anion exchange chromatography may be performed using a common anionexchange resin. Examples of the anion exchange resin may includeFractogel® EMD TMAE (M), Fractogel® EMD TMAE Medcap (M), Fractogel® EMDTMAE Hicap (M), Eshmuno® Q, Eshmuno® QPX, Eshmuno® QPX Hicap, Capto Q,Capto Q ImpRes, Q Sepharose® FF, Q Sepharose® HP, Q Sepharose® XL,Source® 30Q, Capto® Adhere, Capto® Adhere ImpRes, Poros® 50 HQ, Poros®50 XQ, Poros® 50 PI, Q HyperCel, Toyopearl® GigaCap Q 650-M, Toyopearl®GigaCap Q 650-S, Toyopearl® Super Q, YMC® BioPro Q, Macro-Prep® High Q,Nuvia® Q, UNOsphere® Q, etc., but are not limited thereto.Alternatively, a weak anion exchange resin with diethylaminoethyl (DEAE)of dimethylaminoethyl (DMAE) functional group may also be used,depending on operating conditions and pI of the protein. Examplesthereof include Fractogel® EMD DEAE, Fractogel® EMD DMAE, Capto® DEAE orDEAE Ceramic HyperD® F.

The anion exchange chromatography may be performed in a bind-and-elutemode, but is not limited thereto.

A loading amount of the anion exchange chromatography may be 10 g/L to50 g/L resin, but is not limited thereto.

An elution buffer of the anion exchange chromatography may includehistidine hydrochloride (His-HCl).

The elution buffer of the anion exchange chromatography may includeabout 1 mM to about 1000 mM, about 10 mM to about 800 mM, about 20 mM toabout 600 mM, about 40 mM to about 400 mM, about 60 mM to about 200 mM,for example, about 100 mM of histidine hydrochloride (His-HCl). Whenhistidine hydrochloride is used in the above concentration range, therecombinant ECM protein may be isolated with excellent purity and yield.

The elution buffer of the anion exchange chromatography may furtherinclude EDTA. A concentration of EDTA may be appropriately selected bythose skilled in the art.

The elution buffer of the anion exchange chromatography may be at pH 4.0to pH 6.0, at pH 4.5 to pH 5.5, for example, at pH 5.0, but is notlimited thereto.

The recombinant ECM protein may be captured by performing the anionexchange chromatography. Therefore, a specific recombinant ECM proteinmay be specifically isolated by performing the anion exchangechromatography.

(2) may further include performing cation exchange chromatography afterperforming the anion exchange chromatography.

The term “cation exchange chromatography (CEX)” is a type of ionexchange chromatography, and uses a cation exchanger having an anionicfunctional group such as a sulfone group, a carboxyl group, etc.

The cation exchange chromatography may include equilibration, sampleloading, washing I, washing II, washing III, and elution.

The cation exchange chromatography may be performed using a commoncation exchange resin. Examples of the cation exchange resin may includeEshmuno® CPS, Eshmuno® CPX, or SP Fast Flow Sepharose®, Eshmuno® SResin, Fractogel® SO3(M), Fractogel SE Hicap (M), SP Cellthru BigBeadPlus®, Streamline® SP, Streamline® SP XL, SP Sepharose® Big Beads,Toyopearl® M-Cap II SP-550EC, SP Sephadex® A-25, Express-Ion® S,Toyopearl® SP-550C, Toyopearl® SP-650C, Source® 30S, Poros® 50 HS,Poros® 50 XS, SP Sepharose® Fast Flow, SP Sepharose® XL, Capto® S,Capto® SP ImRes, Capto® S ImpAct, Nuvia® HR-S,Cellufine® MAX S-r,Cellufine® MAX S-h, Nuvia® S, UNOsphere® S, UNOsphere® Rapid S,Toyopearl® Giga-Cap S-650 (M), S HyperCel Sorbent®, Toyopearl® SP-650M,Macro-Prep® High S, Macro-Prep® CM, S Ceramic HyperD® F, MacroCap® SP,Capto® SP ImpRes, Toyopearl® SP-650S, SP Sepharose® High Perform, Capto®MMC, Capto® MMC Imp Res, Eshmuno® HCX, Nuvia® High c-Prime, etc., butare not limited thereto. Alternatively, a weak cation exchange resin,for example, Fractogel® EMD COO (M), CM Sepharose® HP, CM Sepharose® FF,Toyopearl® AF Carboxy 650-M, Macro-Prep® CM, Toyopearl® GigaCap CM, CMCeramic Hyper® D, or Bio-Rex® 70 may also be used, depending onoperating conditions and pI of the protein.

The cation exchange chromatography may be performed in a bind-and-elutemode, but is not limited thereto.

A loading amount of the cation exchange chromatography may be 10 g/L to50 g/L resin, but is not limited thereto.

In the washing II of the cation exchange chromatography, a washingbuffer II may include about 1 mM to about 1000 mM, about 5 mM to about800 mM, about 10 mM to about 400 mM, about 25 mM to about 200 mM, orabout 50 mM to about 150 mM, for example, about 100 mM of sodiumchloride (NaCl).

In the washing III of the cation exchange chromatography, a washingbuffer III may include about 150 mM to about 500 mM, about 150 mM toabout 400 mM, about 200 mM to about 500 mM, about 200 mM to about 400mM, about 300 mM to about 500 mM, or about 300 mM to about 400 mM, forexample, about 350 mM of sodium chloride (NaCl).

The washing buffer II or III of the cation exchange chromatography mayfurther include Tris-HCl, NaAc, EDTA, or a combination thereof.

The washing buffer II or III of the cation exchange chromatography maybe at pH 5.0 to 8.5, for example, at pH 8.0 or pH 5.5, but is notlimited thereto.

An elution buffer of the cation exchange chromatography may includeabout 50 mM to about 1000 mM, about 100 mM to about 800 mM, about 200 mMto about 600 mM, about 300 mM to about 500 mM, for example about, 370 mMof sodium chloride (NaCl). A concentration of sodium chloride may beappropriately selected in consideration of a balance between purity andyield of the product within the above range.

The elution buffer of the cation exchange chromatography may furtherinclude Tris-HCl, EDTA, or a combination thereof.

The elution buffer of the cation exchange chromatography may be at pH7.5 to pH 8.5, for example, at pH 8.0, but is not limited thereto.

By performing the cation exchange chromatography, protein aggregates,host cell proteins (HCPs), and other impurities may be removed.

The term “aggregates” refers to a form in which several substances arecombined together. The “protein aggregates” refers to a form in whichproteins are accumulated or assembled, and includes aggregates ofabnormal proteins as well as aggregates of normal proteins. The proteinaggregates include those in which target proteins are bound togetherwith other proteins, but there is a difference in that multimericproteins refer to those in which target proteins are bound to eachother.

The term “host cell proteins (HCPs)” refers to process-related proteinimpurities produced by a host organism during preparation and productionof biotherapeutic agents.

(2) may further include performing mixed mode chromatography afterperforming the cation exchange chromatography.

The term “mixed-mode chromatography (MMC)” refers to a chromatographicmethod utilizing one or more types of interactions between a stationaryphase and an analyte.

The mixed mode chromatography may include pre-equilibration,equilibration, sample loading, washing I, washing II, and elution.

The mixed mode chromatography may be performed using a common mixed moderesin. Examples of the mixed mode resin may include Capto ® adhere,etc., but are not limited thereto.

The mixed mode chromatography may be performed in a bind-and-elute mode,but is not limited thereto.

A loading amount of the mixed mode chromatography may be 10 g/L resin to15 g/L resin, but is not limited thereto.

In the washing II of the mixed mode chromatography, a washing buffer IImay include about 200 mM to about 400 mM, about 200 mM to about 350 mM,or about 250 mM to about 350 mM, for example, about 200 mM or about 300mM of arginine.

The washing buffer II of the mixed mode chromatography may furtherinclude Tris-HCl, EDTA, or a combination thereof.

The washing buffer II of the mixed mode chromatography may be at pH 8.5to 9.5, for example, at pH 9.0, but is not limited thereto.

The elution buffer of the mixed mode chromatography may include about100 mM to about 1000 mM, about 200 mM to about 800 mM, about 300 mM toabout 700 mM, about 400 mM to about 600 mM, for example, about 500 mM ofarginine. A concentration of arginine may be appropriately selected inconsideration of a balance between purity and yield of the productwithin the above range.

The elution buffer of the mixed mode chromatography may further includeTris-HCl, EDTA, or a combination thereof.

The elution buffer of the mixed mode chromatography may be at pH 7.5 topH 8.5, for example, at pH 8.0, but is not limited thereto.

By performing the mixed mode chromatography, protein aggregates and HCPsmay be removed.

(2) may further include performing hydrophobic interactionchromatography after performing the mixed mode chromatography.

The term “hydrophobic interaction chromatography (HIC)” refers to achromatographic method utilizing hydrophobic interactions between afunctional group of a stationary phase and an analyte.

The hydrophobic interaction chromatography may include equilibration,sample loading, washing I, washing II, washing III, and elution.

The hydrophobic interaction chromatography may be performed using acommon hydrophobic interaction resin. Examples of the hydrophobicinteraction resin may include Butyl-S Sepharose 6 Fast Flow, CaptoOctyl, Octyl Sepharose 4 Fast Flow, Phenyl Sepharose 6 Fast Flow (lowsub), Capto Butyl, Butyl Sepharose 4 Fast Flow, Phenyl Sepharose HighPerformance, Capto Phenyl ImpRes, Butyl Sepharose High Performance,Capto Butyl ImpRes, Phenyl Sepharose 6 Fast Flow (high sub), CaptoPhenyl (high sub), etc., but are not limited thereto.

The hydrophobic interaction chromatography may be performed in abind-and-elute mode, but is not limited thereto.

A loading amount of the hydrophobic interaction chromatography may be 3g/L to 6 g/L resin.

In the washing II of the hydrophobic interaction chromatography, awashing buffer II may include about 0.1 M to about 1.0 M, about 0.1 M toabout 0.8 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M,about 0.2 M to about 1.0 M, about 0.2 M to about 0.8 M, about 0.2 M toabout 0.6 M, about 0.2 M to about 0.4 M, about 0.3 M to about 1.0 M,about 0.3 M to about 0.8 M, about 0.3 M to about 0.6 M, or about 0.3 Mto about 0.5 M, for example, about 0.4 M of ammonium sulfate.

The washing buffer II of the hydrophobic interaction chromatography mayfurther include Tris-HCl, EDTA, or a combination thereof.

The washing buffer II of the hydrophobic interaction chromatography maybe at pH 7.5 to pH 8.5, for example, at pH 8.0, but is not limitedthereto.

In the washing III of the hydrophobic interaction chromatography, awashing buffer III may include about 0.5 M to about 2.0 M, about 0.5 Mto about 1.8 M, about 1.0 M to about 2.0 M, about 1.0 M to about 1.8 M,about 1.2 M to about 2.0 M, or about 1.2 M to about 1.8 M, for example,about 1.5 M of sodium chloride.

The washing buffer III of the hydrophobic interaction chromatography mayfurther include Tris-HCl, EDTA, or a combination thereof.

The washing buffer III of the hydrophobic interaction chromatography maybe at pH 7.5 to pH 8.5, for example, at pH 8.0, but is not limitedthereto.

An elution buffer of the hydrophobic interaction chromatography mayinclude about 0.1 M to about 1.5 M, about 0.1 M to about 1.2 M, about0.1 M to about 1.0 M, about 0.1 M to about 0.8 M, about 0.3 M to about1.5 M, about 0.3 M to about 1.2 M, about 0.3 M to about 1.0 M, or about0.3 M to about 0.8 M, for example, about 0.5 M of sodium chloride(NaCl). When the concentration of sodium chloride exceeds 1.5 M, therecombinant ECM protein may not be eluted.

The elution buffer of the hydrophobic interaction chromatography mayfurther include Tris-HCl.

The elution buffer of the hydrophobic interaction chromatography may beat pH 7.5 to 8.5, for example, at pH 8.0, but is not limited thereto.

By performing the hydrophobic interaction chromatography, proteinmultimers and HCPs may be removed.

(2) may sequentially include performing anion exchange chromatography;performing cation exchange chromatography; performing mixed modechromatography; and performing hydrophobic interaction chromatography.(2) may further include a known method capable of isolating andpurifying the recombinant protein. For example, harvest andclarification, ultrafiltration, diafiltration, solvent/detergent (S/D)virus inactivation, intermediate depth filtration, or a combination oftwo or more thereof may be further performed. The harvest andclarification, ultrafiltration, diafiltration, S/D virus inactivation,and intermediate depth filtration may be performed according to commonmethods.

(2) may sequentially include performing harvest and clarification of theculture medium obtained in (1); performing ultrafiltration anddiafiltration; performing anion exchange chromatography; performing S/Dvirus inactivation; performing cation exchange chromatography;performing mixed mode chromatography; performing hydrophobic interactionchromatography; performing ultrafiltration and diafiltration; andperforming intermediate depth filtration.

Still another aspect provides a method of assaying the monomer of therecombinant ECM protein, the method including

-   performing size exclusion chromatography on a sample including the    recombinant ECM protein using a mobile phase containing    hydrochloride; and-   analyzing the monomer of the recombinant ECM protein in the sample,    based on the result of the size exclusion chromatography.

The method of assaying the monomer of the recombinant ECM protein may bea method of assaying the monomer of the recombinant HAPLN protein, morespecifically, a method of assaying the monomer of the recombinant HAPLN1protein, and most specifically, a method of assaying the monomer of therecombinant human HAPLN1 protein.

When hydrochloride is included in the mobile phase, the ability toisolate the recombinant ECM protein may be increased and inaccuratepeaks in the chromatogram may be remarkably reduced.

The hydrochloride included in the mobile phase is not limited to itstype. The hydrochloride may be arginine hydrochloride (Arg-HCl), anilinehydrochloride, adenine hydrochloride, guanine hydrochloride, guanidinehydrochloride (Gdn-HCl), histidine hydrochloride (His-HCl), or lysinehydrochloride (Lys-HCl), but is not limited thereto.

The mobile phase may include the hydrochloride at a concentration ofmore than about 0.5 M, about 0.8 M or more, about 1.0 M or more, morethan about 0.5 M to about 10.0 M, more than about 0.5 M to about 8.0 M,more than about 0.5 M to about 4.0 M, about 0.8 M to about 4.0 M, about0.8 M to about 3.0 M, about 0.8 M to about 2.0 M, about 0.8 M to about1.5 M, about 0.8 M to about 1.2 M, about 1.0 M or more to about 10.0 M,about 1.0 M or more to about 8.0 M, about 1.0 M or more to about 4.0 M,about 1.0 M or more to about 3.0 M, or about 1.0 M to about 2.0 M Whenthe concentration of hydrochloride is 0.5 M or less, the ability toisolate the recombinant ECM protein may be decreased. When theconcentration of hydrochloride is 0.5 M or less, the ability to assaythe monomer of the recombinant ECM protein may be decreased. In oneexemplary embodiment, it was confirmed that when hydrochloride is usedas an additive for the mobile phase, the monomer of the recombinant ECMprotein may be accurately analyzed even at a low concentration of about1.0 M.

The term “size exclusion chromatography (SEC)”, which is also referredto as “gel filtration chromatography”, is a method of separatingproteins according to their size. There is no attractive force between astationary phase and a solute, and a mobile phase simply passes throughthe porous stationary phase, unlike other types of chromatography.

The size exclusion chromatography may be performed according to a commonmethod. The size exclusion chromatography may be size exclusionchromatography for analysis.

Since the method assays the monomer of the recombinant ECM protein, itmay accurately analyze ratios of the monomer of the recombinant ECMprotein and other impurities (e.g., multimers, etc.) in the productsafter performing each isolation and/or purification during the processof producing the recombinant ECM protein. Accordingly, the ratio of themonomer of the recombinant ECM protein may be analyzed. Therefore, inthe analysis, it is possible to analyze the ratios of the monomer of therecombinant ECM protein and other impurities.

The method of assaying the monomer of the recombinant ECM protein mayanalyze a ratio of the monomer of the recombinant HAPLN protein, morespecifically, a ratio of the monomer of the recombinant HAPLN1 protein,and most specifically, a ratio of the monomer of the recombinant humanHAPLN1 protein.

Advantageous Effects of Invention

According to a medium composition according to an aspect, animal cellsproducing a recombinant ECM protein may be cultured in a large amount.

According to a method of producing a recombinant ECM protein accordingto another aspect, the recombinant ECM protein may be isolated with highpurity and a monomer of a specific recombinant ECM protein may bespecifically isolated.

According to a method of assaying a monomer of the recombinant ECMprotein according to still another aspect, the monomer of therecombinant ECM protein may be analyzed with high accuracy, and thusratios of the monomer of the recombinant ECM protein and otherimpurities may be analyzed.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a chromatogram of a linear gradient elution of anionexchange chromatography (AEX);

FIG. 2 shows a chromatogram of a step-wise elution of cation exchangechromatography (CEX);

FIG. 3 shows a result of SDS_PAGE_NR of the step-wise elution of CEX;

FIG. 4 shows a chromatogram of a step-wise elution of mixed-modechromatography (MMC);

FIG. 5 shows a result of SDS_PAGE_NR of the step-wise elution of MMC;

FIGS. 6A and 6B show a chromatogram of hydrophobic interactionchromatography (HIC) comparative elution;

FIG. 7 shows a result of SDS_PAGE_NR of HIC comparative elution;

FIG. 8 shows a chromatogram showing a result of performing sizeexclusion chromatography (SEC) analysis for a sample including rhHAPLN1using phosphate buffer(PB)+NaCl, 5 mM EDTA, or 5 mM EDTA+4 M Gdn-HCl asa mobile phase;

FIG. 9 shows a chromatogram showing a result of performing SEC analysisfor a sample including rhHAPLN1 using 50 mM PB+150 mM NaCl+1 M Arg-HCl(pH 6.3) as a mobile phase;

FIG. 10 shows a chromatogram showing a result of performing SEC analysisfor Sample 3 using 50 mM PB+300 mM NaCl, 0.1 M Arg-HCl, 0.5 M Arg-HCl,1.0 M Arg-HCl, or 1.0 M Gdn-HCl as a mobile phase;

FIG. 11 shows a chromatogram showing a result of performing SEC analysisfor Sample 3 using 50 mM PB+300 mM NaCl, 0.1 M urea, 0.5 M urea, 1.0 Murea, 2.0 M urea, 4.0 M urea, or 6.0 M urea as a mobile phase; and

FIG. 12 shows a chromatogram showing a result of performing SEC analysisfor Sample 3 using 1.0 M Gdn-HCl, 4.0 M urea, or 1.0 M Arg-HCl as amobile phase.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present disclosure will be described in more detailwith reference to exemplary embodiments. However, these exemplaryembodiments are only for illustrating the present disclosure, and thescope of the present disclosure is not limited to these exemplaryembodiments.

Example 1: Culture of Cells Producing Recombinant ECM Protein

A vector including a polynucleotide encoding a human HAPLN1 proteinamong ECM proteins was inserted to CHO-K1 cells to prepare a CHO-K1 cellline producing the recombinant human HAPLN1 protein. A cell line showingexcellent protein production amount and quality was selected as a mastercell bank (MCB).

The MCB was sub-cultured and inoculated at a concentration of0.40±0.05×10⁶ cells/mL in a 250 L Hyperforma SUB bioreactor (Thermo),followed by fed-batch culture.

22.36 g of ActiPro™ medium + 0.5846 g of glutamine + 10.00 g of HTSupplement (Thermo Fisher Scientific) + 4.29 g of 10 N NaOH + 1.80 g ofNaHCO₃ were used as a basic medium. A culture temperature, dissolvedoxygen (DO), and pH were set to 36.5° C., 40.0%, and 7.00 ± 0.20,respectively. 1 M sodium carbonate monohydrate was used as a pH controlsolution.

181.04 g of HyClone™ Cell Boost 7a + 12.28 g of FM020a of 10 N NaOH, and94.60 g of HyClone™ Cell Boost 7b + 105.93 g of FM020b of 10 N NaOH wereused as a feeding medium (FM). A feeding strategy is shown as in Table 1below.

TABLE 1 Fed-batch culture Feeding medium Day 3 Day 5 Day 6 Day 8 Day 10FM020a (%(w/w)) 3 5 3 3 3 FM020b (%(w/w)) 0.3 0.5 0.3 0.3 0.3

As a feed additive, 50 µM CuSO₄ was used, which was added to thebioreactor on day 0 of fed-batch culture.

A Glucose feed stock was prepared at 400 g glucose/kg. From day 3 to day13 of fed-batch culture, when the glucose concentration dropped to lessthan 5.0 g/L, glucose was fed in such a way that it was raised to 6.0g/L.

When a viable cell density (VCD) reached 20.00×10⁶ cells/mL, thetemperature was changed to 31.0° C. Cells were harvested on 12 day offed-batch culture or when viability dropped to less than 60%.

Experimental Example 1: Effect of Reducing Formation of Multimer ofRecombinant ECM Protein According to Type of Additive

An experiment was performed to examine an effect of reducing formationof protein multimers according to the type of additive when cellsproducing the recombinant human HAPLN1 protein were cultured.

In detail, cells were cultured in the same manner as in Example 1,except for varying the type, concentration, and feeding strategy of thefeeding additive. Results of cell culture and protein productionaccording to the type and concentration of the feeding additive, andfeeding strategy thereof in each experimental group are shown in Table 2below.

TABLE 2 Experimental group Type of additive∗ Concentration of additiveFeeding strategy Titer (g/L) Qp (pg/cell/day) SEC_LC (Main)(%)Comparative Example 1 - - - 2.66 21.08 51.1 Comparative Example 2 DMSO0.5%(w/v) Feeding on days 4, 6, 8 2.22 18.01 44.8 Comparative Example 3DMSO 0.2%(w/v) Feeding on days 0,3,5,7,9 2.42 19.15 44.4 ComparativeExample 4 Glycerol 1.0%(w/v) Feeding on day 5 2.46 20.46 43.5Comparative Example 5 Poloxamer 188 0.2%(w/v) Feeding on day 5 2.7122.49 47.0 Comparative Example 6 EDTA 1 mM Feeding on day 5 2.03 20.9657.5 Comparative Example 7 PS80 0.04%(w/v) Feeding on day 5 2.75 23.7743.8 Comparative Example 8 Cysteine 1 mM Feeding on days 5, 7, 9 1.9115.83 31.4 Comparative Example 9 GSH 0.2 mM Feeding on days 6, 8 2.3619.48 43.5 Comparative Example 10 GSH and GSSG 0.2 mM and 0.2 mM Feedingon days 6, 8 2.52 20.31 47.0 Comparative Example 11 EDTA and MgCl₂ 1 mMand 50 µM Feeding on day 5 2.16 22.34 - Example 1 CuSO₄ 50 µM Feeding onday 0 3.87 28.77 53.2 ^(∗)DMSO: dimethyl sulfoxide, PS80: polysorbate80, GSH: glutathione, GSSG: glu-tathione disulfide.

As shown in Table 2, when CuSO₄ additive was used, a titer of therecombinant human HAPLN1 protein production was 3.87 g/L, which was thehighest, and the production amount of the recombinant human HAPLN1protein per cell was 28.77 pg/ cell/day, which was the highest.

Therefore, it was confirmed that when a copper compound such as CuSO₄ isused as an additive during culturing cells producing the recombinanthuman HAPLN1 protein, the effect of reducing formation of the proteinmultimer is excellent and the protein production amount is increased.Specifically, when CuSO₄ at a concentration of 50 µM was fed on day 0 ofcell fed-batch culture, the highest effect of reducing formation of theprotein multimer and the highest protein production amount wereobserved. Accordingly, it is suggested that CuSO₄ may be used inmass-production of the recombinant human HAPLN1 protein.

Experimental Example 2: Effect of Reducing Formation of Recombinant ECMProtein Multimer According to Concentration of Copper Compound

An experiment was performed to examine an effect of reducing formationof protein multimers according to concentrations of the copper compoundwhen cells producing the recombinant human HAPLN1 protein were cultured.

In detail, cells were cultured in the same manner as in Example 1,except for varying the concentration of the copper compound. Theconcentration of the copper compound in each experimental group andresults of protein production according to the concentration are shownin Table 3 below.

TABLE 3 Experimental group CuSO₄ concentration Titer (D10) (g/L) SECCaliper_NR Main Peak % HMW Peak % LMW Peak % Purity % peak1 % Peak2 %Peak3 % Example 1 50 µM 2.57 50.3 38.5 11.2 85.7 20.6 65.1 0.68Comparative Example 12 20 µM 2.42 51.8 38.8 9.4 86.7 20.9 65.8 0.67Comparative Example 13 5 µM 2.42 51.3 38.8 9.9 86.0 20.6 65.4 0.71

As shown in Table 3, when CuSO₄ was used at a concentration of more than20 µM, a titer of the human HAPLN1 protein production was increased. Inparticular, Example 1, in which CuSO₄ was used at a concentration of 50µM or more, showed the excellent titer of the human HAPLN1 proteinproduction. Therefore, when a copper compound such as CuSO₄ is used at aconcentration of more than 20 µM, particularly, 50 µM or more duringculturing the cells producing the recombinant human HAPLN1 protein, theexcellent effect of reducing formation of the protein multimer and theincreased protein production amount were observed.

Example 2: Isolation and Purification of Recombinant ECM Protein

The recombinant human HAPLN1 protein was isolated and purified from thecells cultured according to Example 1. In detail, isolation andpurification of the recombinant human HAPLN1 protein were performedaccording to the following procedures.

Harvest and Clarification

For harvest and clarification, DOHC and A1HC depth filter of Milliporewere used. Recommended loading volumes of DOHC and A1HC depth filtersare 45 L/m² and 90 L/m², respectively.

Ultrafiltration/Diafiltration 1 (UF/DF1)

Pellicon 3 (Ultracel, Type C Screen, 30 kDa) of Millipore was selectedas a UF/DF1 membrane. A concentration of the loading sample was 5 g/L orless in the UF, and then diafiltration was performed with 6 times ormore volume of a buffer containing 50 mM Tris-HCl and 5 mM EDTA at pH9.0. A feed flow rate was 300 LMH or less and a transmembrane pressure(TMP) was 10 psi to 20 psi. A recommended loading amount is 70 L/m² orless.

Anion Exchange Chromatography (AEX)

Poros 50HQ resin of Life Tech was used as a capture resin. This step wasperformed in a bind-and-elute mode. A recommended protein loading amountis 10 g/L resin to 50 g/L resin. A selected elution buffer is a buffercontaining 100 mM His—HCl and 5 mM EDTA at pH 5.0. A recommended UV peakcollection range is 25 mAU/mm to 75 mAU/mm.

Solvent/Detergent (S/D) Virus Inactivation

S/D virus inactivation was performed according to a common method.

Cation Exchange Chromatography (CEX)

This step was performed in a bind-and-elute mode. Capto S ImpAct resinof Cytiva (formerly GE Healthcare) was used as a CEX resin. Arecommended CEX loading amount is 10 g/L resin to 15 g/L resin. Byperforming CEX, protein aggregates, host cell proteins (HCPs), and otherimpurities were removed. A buffer containing 50 mM Tris-HCl, 100 mMNaCl, and 5 mM EDTA at pH 8.0 is recommended as a washing buffer II, anda buffer containing 50 mM NaAc, 350 mM NaCl, and 5 mM EDTA at pH 5.5 isrecommended as a washing buffer III. A recommended elution buffer is abuffer containing 50 mM Tris-HCl, 370 mM NaCl, and 5 mM EDTA at pH 8.0.A recommended UV peak collection range is 25 mAU/mm to 50 mAU/mm.

Mixed-Mode Chromatography (MMC)

This step was performed in a bind-and-elute mode. Capto adhere resin ofCytiva (formerly GE Healthcare) was used as an MMC resin. A recommendedloading amount is 10 g/L resin to 15 g/L resin. To further removeprotein aggregates and HCPs, a buffer containing 50 mM Tris-HCl, 0.5 Marginine(Arg), and 5 mM EDTA at pH 8.0 was used as an elution buffer.

Hydrophobic Interaction Chromatography (HIC)

This step was performed in a bind-and-elute mode. Butyl Sepharose 4 FastFlow resin of Cytiva (formerly GE Healthcare) was used as an HIC resin.A recommended loading amount is 3 g/L resin to 6 g/L resin. A buffercontaining 50 mM Tris-HCl and 1.5 M NaCl at pH 8.0 is recommended as awashing buffer III. The target protein was eluted with high purity usinga buffer containing 50 mM Tris-HCl and 0.5 M NaCl at pH 8.0.

Ultrafiltration/Diafiltration 2 (UF/DF2)

Pellicon 3 (Ultracel, Type C Screen, 10 kDa) of Millipore was selectedfor UF/DF2. The loading sample was concentrated to 1 g/L to 3 g/L in UF,and subjected to diafiltration with 6 times or more volume of a buffercontaining 20 mM NaAc at pH 5.0. A UF/DF2 pool concentration was 4.5mg/mL to 5.5 mg/mL. A feed flow rate was 300 LMH or less, and atransmembrane pressure (TMP) was 10 psi to 20 psi. A load capacity is 70g/m² or less.

Intermediate Depth Filtration (Int. DF)

For intermediate depth filtration, X0SP depth filter of Millipore wasselected to remove HCPs. A recommended loading amount of the X0SP filteris 400 g/m² to 800 g/m².

Formulation and Bulk Fill

A concentration of a drug substance (DS) is 2.0±0.2 g/L. A compositionof a formulation buffer was determined by drug product development (DPD)and transferred to downstream process development (DSPD). PS80 andsucrose were added to VF pool samples at final concentrations of 0.04%(w/v) and 8% (w/v), respectively. DS was obtained after 0.2 µm finalfiltration.

Experimental Example 3: AEX Conditions for Specifically IsolatingSpecific Recombinant ECM Protein

Among ECM proteins, HAPLN1 protein has a molecular weight of 40 kDa to50 kDa.

In the anion exchange chromatography (AEX) of Example 2-(3), anexperiment was performed to optimize AEX conditions for specificallyisolating a recombinant human HAPLN1 protein.

Elution Conditions (1.1) AEX Linear Gradient Comparative ElutionMaterials

-   Column: Poros 50HQ, 3.024 mL (0.5 cm X 15.4 cm)-   Loading material: concentration 2.64 g/L, pH 9.06, conductivity 1.44    mS/cm-   Loading amount: 30 g/L resin-   Column washing (sanitization) solution: 1.0 M NaOH-   Pre-equilibration buffer: 50 mM Tris-HCl, 1 M (NH₄)₂SO₄, 5 mM EDTA,    pH 8.0-   Equilibration/washing buffer I: 50 mM Tris-HCl, 50 mM NaCl, 5 mM    EDTA, pH 9.0-   Washing buffer II: 50 mM Tris-HCl, 10 mM NaCl, 5 mM EDTA, pH 9.0-   Elution buffer: control: 50 mM Tris-HCl, 80 mM (NH₄)₂SO₄,5 mM EDTA,    pH 8.5; condition 1-A: 20 mM His-HCl, pH 7.5, B: 50 mM His-HCl, 30    mM NaCl, 5 mM EDTA, pH 5.8; condition 2-A: 100 mM His-HCl, pH 7.0,    B: 100 mM His-HCl, 5 mM EDTA, pH 5.5; condition 3-A: 100 mM His-HCl,    pH 7.0, B: 100 mM His-HCl, 5 mM EDTA, pH 5.0; linear gradient    elution from A to B for 20 column volumes (CV)-   Stripping buffer: 50 mM Tris-HCl, 1 M (NH₄)₂SO₄, 5 mM EDTA, pH 8.0-   Column storage solution: 20% ethanol

Experimental Procedure

Eluates were collected at 25 mAU/mm to 25 mAU/mm. The elution conditionswere verified using a chromatogram.

Results

A yield of the recombinant human HAPLN1 protein, SEC analysis results,and HCP concentrations according to each elution condition are shown inTable 4 below. As shown in Table 4, when 100 mM His-HCl was addedwithout salts to the elution buffer, better effect of capturing therecombinant human HAPLN1 protein was observed, as compared with thosewith salts. In addition, when 100 mM His-HCl was added, HCPs weredecreased by 50%, as compared with a control.

TABLE 4 Run Washing buffer II Elution conditions (linear gradientelution from A to B) Yield* (%) SEC(%) M/H/L HCP (ppm ) A B 1(Control) - - 50 mM Tris-HCl, 80 mM (NH₄)₂SO₄, 5 mM EDTA, pH8.5 7953.1/46.7/0. 2 1646 08 2 (Elution condition 1) 50 mM Tris-HCl,10 mMNaCl, 5 mM EDTA, pH9.0 20 mM His-HCl, pH7.5 50 mM His-HCl, 30 mM NaCl, 5mM EDTA, pH5.8 74 56.7/43.3/N D 7311 2 3 (Elution condition 2) 100 mMHis-HCl, pH7.0 100 mM His-HCl, 5 mM EDTA, pH5.5 78 55.8/44.2/N D 7985 64 (Elution condition 3) 100 mM His-HCl, 5 mM EDTA, pH5.0 85 54.4/45.6/ND 8365 4

(1.2) AEX Linear Gradient Elution Materials

-   Column: Poros 50HQ, 3.024 mL (0.5 cm X 15.4 cm)-   Loading material: concentration 2.64 mg/mL, pH 9.06, conductivity    1.44 mS/cm-   Loading amount: 30 g/L resin-   Sanitization solution: 1.0 M NaOH-   Pre-equilibration buffer: 50 mM Tris-HCl, 1 M (NH₄)₂SO₄, 5 mM EDTA,    pH 8.0-   Equilibration/washing buffer I: 50 mM Tris-HCl, 50 mM NaCl, 5 mM    EDTA, pH 9.0-   Washing buffer II: 50 mM Tris-HCl, 5 mM EDTA, pH 9.0-   Elution buffer: A: 100 mM His-HCl, pH 7.0, B: 100 mM His-HCl, 5 mM    EDTA, pH 5.0; linear gradient elution from A to B for 20 column    volumes (CV).-   Stripping buffer: 50 mM Tris-HCl, 450 mM (NH₄)₂SO₄, 5 mM EDTA, pH    8.0-   Storage solution: 20% ethanol

Experimental Procedure

Based on the results of linear gradient comparative elution, thematerial after UF/DF1 was loaded into a Poros 50HQ column, and anoptimal elution buffer was determined using a step-wise elution method.Optimal elution conditions were confirmed using SEC purity analysis.

Results

FIG. 1 shows a chromatogram of the linear gradient elution of AEX.

As shown in Table 4 and FIG. 1 , 100 mM His-HCl efficiently captured therecombinant human HAPLN1 protein without additional salt addition. For abalance between purity and yield, pH 5.0 was chosen as pH for elution.

Therefore, it was found that about 100 mM His-HCl may be selected as theAEX elution buffer in order to specifically isolate the recombinanthuman HAPLN1 protein. In particular, when 100 mM His-HCl is used, therecombinant human HAPLN1 protein may be isolated with excellent purityand yield. For example, a buffer containing 100 mM His-HCl and 5 mM EDTAat pH 5.0 may be used as the AEX elution buffer.

Experimental Example 4: CEX Conditions for Removing Recombinant ECMProtein Aggregates, HCPs, and Other Impurities

The bind-and-elute mode CEX of Example 2-(5) was introduced to removerecombinant human HAPLN1 protein aggregates, HCPs, and other impurities.Therefore, an experiment was performed to optimize the CEX conditionsfor removing recombinant human HAPLN1 protein aggregates, HCPs, andother impurities.

Elution Conditions Materials

-   Column: Capto S ImpAct, 2.631 mL (0.5 cm X 13.4 cm)-   Loading material: AEX eluate, concentration 11.844 mg/mL, pH 5.52,    conductivity 11.70 mS/cm-   Loading amount: 10 mg/mL resin-   Equilibration/Washing I buffer: 50 mM NaAc-HAc, 5 mM EDTA, pH 5.5-   Washing II buffer: 50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, pH 8.0-   Washing III buffer: 50 mM NaAC, 350 mM NaCl, 5 mM EDTA, pH 5.5-   Elution buffer: (A) 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; (B) 50 mM    Tris-HCl, 500 mM NaCl, 5 mM EDTA, pH 8.0; step-wise elution: 20% B    (100 mM NaCl), 5 CV; 40% B (200 mM NaCl), 5 CV; 60% B (300 mM NaCl),    5 CV; 75% B (375 mM NaCl), 5 CV; 85% B (425 mM NaCl)-   Stripping buffer: 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, pH 8.0

Experimental Procedure

Optimal elution conditions were confirmed by performing a step-wiseelution method. A loading amount was 10 g/L resin, and eluates werecollected at 25 mAU/ mm to 25 mAU/mm. A protein concentration of eachfraction was measured, and step-wise recovery amounts were calculated.In addition, sample purity was analyzed by SDS_PAGE_NR.

Results

The elution conditions are important for product quality. Criteria forthe optimal conditions are based on the removal of impurities.

FIG. 2 shows a chromatogram of the step-wise elution of CEX.

FIG. 3 shows a result of SDS_PAGE_NR of the step-wise elution of CEX.

The HCP test results in the CEX step-wise elution are shown in Table 5below.

TABLE 5 Fraction NaCl concentration (mM) yield (%) HCP (ng/mg) Load - -121669 F01 0 2.0 - E01 100 0.7 799110 E02 200 20.0 115447 E03 300 32.0E04 375 9.8 35248 E05 425 1.6 - S01 500 9.2 11179

As shown in FIG. 3 and Table 5, when 100 mM NaCl (E01) was used, thelargest amount of HCPs was removed, and loss of the target protein wasonly 0.7%. Yields of fraction E02-E04 and fraction E05 were 61.8% and1.6%, respectively. The HMW content was increased with increasing NaCl.For a balance between purity and yield of the product, a buffercontaining 50 mM Tris-HCl, 370 mM NaCl, 5 mM EDTA at pH 8.0 wasrecommended as the elution buffer.

Experimental Example 5: MMC Conditions for Removing Recombinant ECMProtein Aggregates and HCPs

The bind-and-elute mode MMC of Example 2-(6) was used to additionallyremove recombinant human HAPLN1 protein aggregates and HCPs. Therefore,an experiment was performed to optimize the MMC conditions for removingrecombinant human HAPLN1 protein aggregates and HCPs.

Elution Conditions

The recombinant human HAPLN1 protein aggregates and HCPs were removedusing a capto adhere. CEX eluates were loaded onto the Capto adherecolumn. Based on the result of linear gradient elution for 50 L materialproduction, optimal elution conditions were confirmed by a step-wiseelution method.

Materials

-   Column: Capto adhere, 2.985 mL (0.5 cm X 15.2 cm)-   Loading material: CEX eluate, concentration 3.105 mg/mL, HCP 155217    ng/mg, SEC purity 52.9%-   Loading amount: 7.5 g/L resin-   Pre-equilibration buffer: 50 mM NaAc-HAc, 1 M NaCl, 5 mM EDTA, pH    5.5-   Equilibration/washing buffer I: 50 mM Tris-HCl, 5 mM EDTA, pH 8.0-   Elution buffer: (A) 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; (B) 50 mM    Tris-HCl, 1 M Arg, 5 mM EDTA, pH 8.0; step-wise elution: 20% B (200    mM Arg), 5 CV; 40% B (400 mM Arg), 10 CV; 50% B (500 mM Arg), 10 CV;    60% B (600 mM Arg), 10 CV; 70% B (700 mM Arg), 10 CV-   Stripping buffer: 50 mM HAc

Experimental Procedure

The optimal elution conditions were determined by step-wise elution. CEXeluates were loaded into the Capto adhere column with 7.5 g/L resin, andthe eluates were collected at 25 mAU/mm to 25 mAU/mm. A proteinconcentration of each fraction was measured, and step-wise recoveryamounts were calculated. HCP and SDS_PAGE_NR purity were also tested.

Results

FIG. 4 shows a chromatogram of the step-wise elution of MMC.

FIG. 5 shows a result of SDS_PAGE_NR of the step-wise elution of MMC.

The results of testing HCPs in the MMC step-wise elution are shown inTable 6 below.

TABLE 6 Fraction Arginine concentration (mM) Yield (%) HCP (ng/mg)Load - - 155217 Peak 1 (E01) 200 0.2 2711333 Peak 2 (E02-E03) 400 39.539995 Peak 3 (E04-E05) 500 28.1 4295 Peak 4 (E06) 600 6.6 5865 Peak 5(E07) 700 0.7 - Peak 6 (S01) 1000 1.9 27198

As shown in FIG. 5 and Table 6, when 200 mM arginine (peak 1) was used,the largest amount of HCPs was removed, and loss of the target proteinwas only 0.2%. Yields of peak 2 and peak 3 were 39.5% and 28.1%,respectively. When the arginine concentration was high, the largestamount of HMW was eluted. For a balance between purity and yield of theproduct, a buffer containing 50 mM Tris-HCl, 500 mM arginine, and 5 mMEDTA at pH 8.0 was recommended as the elution buffer. 200 mM argininemay be used in washing.

Experimental Example 6: HIC Conditions for Improving Purity ofReCombinant ECM Protein

The HIC of Example 2-(7) was used to remove recombinant human HAPLN1protein multimers and HCPs. Therefore, an experiment was performed tooptimize the HIC conditions for removing the recombinant human HAPLN1protein multimers and HCPs.

Elution Conditions Materials

-   Column: Butyl Sepharose 4 Fast Flow, 2.631 mL (0.5 cm X 13.4 cm)-   Loading materials: 1) MMC eluate, concentration 0.590 mg/mL, pH    8.09, 146.35 mS/cm, SEC purity 68.9%; 2) MMC eluate, concentration    0.543 mg/mL, pH 8.10, 145.82 mS/cm, SEC purity 68.9%-   Loading amount: 5 g/L resin-   Equilibration/washing buffer I: 50 mM Tris-HCl, 1 M (NH₄)₂SO₄, 5 mM    EDTA, pH 8.0-   Washing buffer II: 50 mM Tris-HCl, 0.4 M (NH₄)₂SO₄, 5 mM EDTA, pH    8.0-   Washing buffer III: 1) 50 mM Tris-HCl, 2 M NaCl, 5 mM EDTA, pH    8.0; 2) 50 mM Tris-HCl, 1.5 M NaCl, 5 mM EDTA, pH 8.0-   Elution buffer:    -   1) (A) 50 mM Tris-HCl, 2 M NaCl, 5 mM EDTA, pH 8.0; (B) 50 mM        Tris-HCl, 5 mM EDTA, pH 8.0; step-wise elution: 25% B (1.5 M        NaCl), 10 CV; 50% B (1 M NaCl), 10 CV; 75% B (0.5 M NaCl), 10        CV; 90% B (0.2 M NaCl), 10 CV; 100% B (0 M NaCl), 10 CV;    -   2) 50 mM Tris-HCl, 0.5 M NaCl, pH 8.0

    - Stripping buffer: 50 mM Tris-HCl, 5 mM EDTA, pH 8.0

Experimental Procedure

Washing III and elution conditions were determined by performing astep-wise elution method. MMC eluates were adjusted with ~1 M (NH₄)₂SO₄before loading on the HIC column. A loading amount was 5 g/L resin, andeluates were collected at 25 mAU/mm to 25 mAU/mm. A proteinconcentration of each fraction was measured, and step-wise recoveryamounts were calculated. Purity was tested using SDS_PAGE_NR.

Results

FIGS. 6A and 6B show a chromatogram of HIC comparative elution.

FIG. 7 shows a result of SDS_PAGE_NR of HIC comparative elution.

The results of the HIC step-wise elution are shown in Table 7 below.

TABLE 7 Run No. Washing III conditions Elution conditions Fraction Yield(%) SEC purity (%) 1) 2 M NaCl Step-wise (1.5 → 1 → 0.5 → 0.2 → 0 MNaCl) Washing 45.8 - Elution 15.3 - 2) 1.5 M NaCl One-step (0.5 M NaCl)Washing 50.1 - Elution 34.8 96.7/3.3/ND

As shown in FIG. 7 and Table 7, the largest amount of HMW was removed bythe washing buffer II, and the target protein was not eluted in thepresence of NaCl exceeding 1.5 M at pH 8.0. Therefore, in Run2, a buffercontaining 50 mM Tris-HCl, 1.5 M NaCl, and 5 mM EDTA at pH 8.0 was usedin Wash III, and a buffer containing 50 mM Tris-HCl, 0.5 M NaCl, and 5mM EDTA at pH 8.0 was used in elution. Finally, a yield of the secondexperiment was 34.8%. Therefore, a buffer containing 50 mM Tris-HCl, 1.5M NaCl, and 5 mM EDTA at pH 8.0 was used as the washing buffer III, anda buffer containing 50 mM Tris-HCl and 0.5 M NaCl at pH 8.0 was used asthe elution buffer.

Example 3: Method of Assaying Recombinant ECM Proteins

The monomers of the recombinant ECM protein and other impurities insamples were analyzed by performing size exclusion chromatography (SEC)using a mobile phase containing hydrochloride. Specific conditions forSEC are as follows:

-   Column: TSKgel G3000SWXL, 7.8 × 300 mm, 5 µm Steel (Manuf. TOSOH)-   Mobile phase: 50 mM phosphate buffer (PB), 300 mM NaCl, 1 M Gdn-HCl    or Arg-HCI pH7.5 (±0.5)-   Detection wavelength: 280 nm-   Flow rate: 1.0 mL/min-   Column temperature: 25 ± 3° C.-   Sample temperature: 5 ± 3° C.-   Sample feeding amount: 100 µg

Experimental Example 7: Screening for Additives for Accurate Analysis ofReCombinant ECM Proteins

An experiment was performed to screen for additives of a mobile phase,which is able to improve accuracy by reducing appearance of inaccuratepeaks, when the monomers of the recombinant ECM protein and otherimpurities are analyzed using SEC. In detail, to analyze the monomers ofthe recombinant human HAPLN1 protein and other impurities using SEC,accuracy of SEC analysis according to the type and concentration of theadditive used as the mobile phase was examined. As a sample, anintermediate product during the isolation and purification of therecombinant human HAPLN1 protein according to Example 2 was used.

FIG. 8 shows a chromatogram showing a result of performing SEC analysisfor a sample including the recombinant human HAPLN1 protein usingphosphate buffer(PB)+NaCl, 5 mM EDTA, or 5 mM EDTA+4 M Gdn-HCl as themobile phase (MP).

FIG. 9 shows a chromatogram showing a result of performing SEC analysisfor a sample including the recombinant human HAPLN1 protein using 50 mMPB+150 mM NaCl+1 M Arg-HCl (pH 6.3) as the mobile phase.

As shown in FIGS. 8 to 9 , when hydrochloride was added to the mobilephase, the monomer peak appeared most clearly.

FIG. 10 shows a chromatogra showing a result of performing SEC analysisusing 50 mM PB+300 mM NaCl, 0.1 M Arg-HCl, 0.5 M Arg-HCl, 1.0 M Arg-HCl,or 1.0 M Gdn-HCl as the mobile phase. The analysis results are shown inTable 8 below.

TABLE 8 Mobile phase Monomer (%) HMW (%) LMW (%) 50 mM PB+300 mM NaCl22.4 76.9 0.8 0.1 M Arg-HCl 26.1 73.9 ND 0.5 M Arg-HCl 62.0 38.0 ND 1.0M Arg-HCl 71.8 28.2 ND 1.0 M Gdn-HCl 77.2 22.8 ND

As shown in FIG. 10 and Table 8, the recombinant human HAPLN1 proteinwas well isolated with increasing concentration of Arg-HCl. Inparticular, when Arg-HCl and Gdn-HCl were used at a concentration of 1.0M, the ability to isolate the recombinant human HAPLN1 protein wasexcellent.

FIG. 11 shows a chromatogram showing a result of performing SEC analysisusing urea, which is known as a monomer assay additive, as a mobilephase, such as 50 mM PB+300 mM NaCl, 0.1 M urea, 0.5 M urea, 1.0 M urea,2.0 M urea, 4.0 M urea, or 6.0 M urea. The analysis results are shown inTable 9 below.

TABLE 9 Mobile phase Monomer (%) HMW (%) LMW (%) 50 mM PB+300 mM NaCl26.4 73.6 ND 0.1 M urea 26.4 73.6 ND 0.5 M urea 26.7 73.3 ND 1.0 M urea27.7 72.3 ND 2.0 M urea 32.1 67.9 ND 4.0 M urea 75.6 24.4 ND 6.0 M urea75.6 24.4 ND

As shown in FIG. 11 and Table 9, the recombinant human HAPLN1 proteinwas well isolated with increasing concentration of urea. However, unlikehydrochloride, when urea was used at a high concentration of 4.0 M ormore, it was possible to isolate the recombinant human HAPLN1 protein.

FIG. 12 shows a chromatogram showing a result of performing SEC analysisusing 1.0 M Gdn-HCl, 4.0 M urea, or 1.0 M Arg-HCl as a mobile phase. Theanalysis results are shown in Table 10 below.

TABLE 10 Mobile phase Monomer (%) HMW (%) LMW (%) 1.0 M Gdn-HCl 77.222.8 ND 4.0 M urea 75.6 24.4 ND 1.0 M Arg-HCl 71.8 28.2 ND

As shown in FIG. 12 and Table 10, when hydrochloride such as Gdn-HCl andArg-HCl is used even at a low concentration of 1.0 M, the ability toisolate the recombinant human HAPLN1 protein was equivalent to or higherthan that of using urea at a high concentration of 4.0 M.

Therefore, when SEC analysis was performed using 1.0 M hydrochloride asthe additive of the mobile phase, the excellent ability to isolate therecombinant human HAPLN1 protein was observed, and inaccurate peaks inthe chromatogram were significantly reduced, allowing accurate analysisof the monomer of the recombinant human HAPLN1 protein.

Taken together, it was confirmed that a ratio of the recombinant ECMprotein monomer in the sample may be analyzed by performing sizeexclusion chromatography using the mobile phase containinghydrochloride.

1. A medium composition for culturing animal cells for producing arecombinant extracellular matrix protein, the medium compositioncomprising a copper compound.
 2. The medium composition of claim 1,wherein the copper compound is copper oxide (Cu₂O), copper chloride(C_(U)C1₂), copper nitrate (Cu(NO₃)₂), copper II oxide (CuO), coppersulfide (CuS), or copper sulfate (CuSO₄).
 3. The medium composition ofclaim 1, comprising the copper compound at a concentration of more than20 µM.
 4. The medium composition of claim 1, wherein the recombinantextracellular matrix protein is a monomer.
 5. The medium composition ofclaim 1, wherein the recombinant extracellular matrix protein iscollagen, elastin, fibronectin, laminin, vitronectin, tenascin, orhyaluronan and proteoglycan link protein (HAPLN).
 6. The mediumcomposition of claim 5, wherein the HAPLN protein is any one proteinselected from the group consisting of HAPLN1, HAPLN2, HAPLN3, andHAPLN4.
 7. The medium composition of claim 1, wherein the recombinantextracellular matrix protein is a protein derived from a human body. 8.The medium composition of claim 1, wherein the animal cells are Chinesehamster ovary (CHO) cells or CHO cell variants.
 9. A method of producinga recombinant extracellular matrix protein with high purity, the methodcomprising: (1) culturing animal cells producing the recombinantextracellular matrix protein, in the medium composition of claim 1, andobtaining a culture medium; and (2) isolating and purifying therecombinant extracellular matrix protein from the culture medium. 10.The method of claim 9, wherein the culturing of (1) is fed-batchculture, continuous culture, or batch culture.
 11. The method of claim9, wherein, in (1), a copper compound is added to the medium compositionbefore culturing the cells.
 12. The method of claim 9, wherein therecombinant extracellular matrix protein is a monomer. 13-16. (canceled)17. The method of claim 9, wherein (2) comprises performingchromatography.
 18. The method of claim 17, wherein the chromatographyis any one or more selected from the group consisting of affinitychromatography, anion exchange chromatography, cation exchangechromatography, hydroxyapatite chromatography, reversed-phasechromatography, size exclusion chromatography, mixed modechromatography, and hydrophobic interaction chromatography.
 19. Themethod of claim 17, wherein the performing of the chromatographycomprises performing anion exchange chromatography; performing cationexchange chromatography; performing mixed mode chromatography; andperforming hydrophobic interaction chromatography.
 20. The method ofclaim 19, wherein an elution buffer of the anion exchange chromatographycomprises 60 mM to 200 mM of histidine hydrochloride (His-HCl).
 21. Themethod of claim 19, wherein an elution buffer of the cation exchangechromatography comprises 200 mM to 600 mM of sodium chloride (NaC1). 22.The method of claim 19, wherein an elution buffer of the mixed modechromatography comprises 200 mM to 800 mM of arginine.
 23. The method ofclaim 19, wherein an elution buffer of the hydrophobic interactionchromatography comprises 0.1 M to 1.5 M of sodium chloride (NaCl). 24.The method of claim 9, wherein the recombinant extracellular matrixprotein isolated and purified in (2) has a purity of 90% or more. 25-30.(canceled)